Appropriate platelet adhesion, activation and aggregation are important in maintaining a balance between normal hemostasis and pathological arterial thrombosis such as stroke and myocardial infarction. Exposure of matrix protein collagen after vessel injury provides a substrate for platelet adhesion and triggers platelet activation, which recruits additional platelets to area of injured vessel wall, thereby initiating thrombus formation. Platelet adhesion and aggregation are critical events in intravascular thrombosis. The formation of a blood clot is normally the result of tissue injury which initiates platelet adhesion/aggregation and coagulation cascade and has the effect of slowing or preventing blood flow in wound healing. However, in certain disease states the formation of blood clots within the circulatory system reaches an undesired extent and is itself the source of morbidity potentially leading to pathological consequences. Activated under conditions of turbulent blood flow in diseased vessels or by the release of mediators from other circulating cells and damaged endothelial cells lining the vessel, platelets accumulate at a site of vessel injury and recruit further platelets into the developing thrombus. The thrombus can grow to sufficient size to block off arterial blood vessels. Thrombi can also form in areas of stasis or slow blood flow in veins. Venous thrombi can easily detach portions of themselves called emboli that travel through the circulatory system and can result in blockade of other vessels, such as pulmonary arteries. Thus, arterial thrombi cause serious disease by local blockade, whereas venous thrombi do so primarily by distant blockade, or embolization. These conditions include venous thrombosis, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, cerebral embolism, kidney embolisms and pulmonary embolisms.
Many adhesive proteins and various receptors are involved in the complex progress of platelet adhesion, activation and aggregation. Circulating platelets become adherent and form an occlusive thrombus either by exposure to atherosclerotic lesions following plaque rupture or in response to pathological shear stress. Integrin α2β1 [glycoprotein (GP) Ia/IIa] and GPVI are two major platelet receptors for collagen, and mediate platelet adhesion and aggregation. Under high shear conditions, GPIb-V-IX complex is considered to be an indirect collagen receptor, acting through the binding of von Willebrand factor.
Snake venoms contain many biological components that affect hemostasis by various mechanisms, including affecting platelet function or coagulation factors, or disrupting endothelium. U.S. Pat. No. 6,284,475 uses this characteristic and provides methods for diagnosing and/or monitoring thrombophilic disease in a patient that can result from the antiphospholipid antibody syndrome (aPL syndrome), which are premised on the inhibition of binding of an anticoagulant protein, annexin, preferably annexin-V, to phospholipids by antiphospholipid (aPL) antibodies in a patient blood sample. C-type lectin-like proteins (CLPs) composed of αβ heterodimers are an important family in snake venoms. CLPs are often oligomerized to form large molecules and interact with specific platelet receptors such as GPIb, α2β1 or/and GPVI to activate or inhibit platelet function. Convulxin (CVX), a multimeric protein from Crotalus durissus terrificus venom, induces platelet activation via binding to GPVI and GPIb. The molecular interaction between convulxin and GPVI has been examined by X-ray crystallography, and the putative GPVI-binding sites of convulxin have been studied (Batuwangala T, Leduc M, Gibbins J M, et al. Structure of the snake-venom toxin convulxin. Acta crystallographica 2004 January; 60(Pt 1):46-53). Aggretin, also known as rhodocytin, purified from Calloselasma rhodostoma venom, has been shown to bind to α2β1, GPIb, and CLEC-2. Recently, the crystal structure of aggretin has been studied and its binding sites also have been proposed (Hooley E, Papagrigoriou E, Navdaev A, et al. The crystal structure of the platelet activator aggretin reveals a novel (alphabeta) 2 dimeric structure. Biochemistry 2008 Jul. 29; 47(30):7831-7; Watson A A, Eble J A, O'Callaghan C A. Crystal structure of rhodocytin, a ligand for the platelet-activating receptor CLEC-2. Protein Sci 2008 September; 17(9):1611-6). On the other hand, agkistin, purified from Agkistrodon acutus venom, inhibits platelet aggregation through its specific binding to platelet GPIb (Yeh C H, Chang M C, Peng H C, et al. Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist. British journal of pharmacology 2001 February; 132(4):843-5013). Trowaglerix, a CLPs purified from Tropidolaemus waglerix venom, specifically activates platelets via GPVI (Chang C H, Chung C H, Kuo H L, et al. The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding. J Thromb Haemost 2008 April; 6(4):669-76). In a previous study, trowaglerix caused GPVI cleavage in vitro and abolished collagen-induced aggregation ex vivo. While the structure of CLPs together with their ligands have been determined, casting some light on the potential binding sites in a molecular level, the linear binding motif is also a very helpful indication for these binding ligands.
However, although the structure of CLPs together with their ligands have been determined, there is still a need to cast some light on the binding sites in a molecular level sp as to develop usable drugs with advantageous platelet aggregation inhibitory effect and antithrombotic activity without hemorrhagic tendency.